Upon interacting with APCs, T cells assemble a signaling complex termed the Immunological Synapse(IS) at the cell-cell contact site, and a second protein complex termed the Distal Pole Complex (DPC) at the opposite pole. The DPC remains poorly understood, but it is believed to sequester negative regulators of T cell activation. Formation of the DPC is dependent on the ERM proteins ezrin and moesin; disruption of ERM function disperses the complex and inhibits T cell activation. Among the DPC proteins identified to date are signaling molecules such as SHP-1 and PI3K, as well as Discs Large (hDLG) and scribble, proteins that affect T cell signaling and control cell polarity in other cell types. In this project, we will test the hypothesis that the DPC serves a dual function: to organize T cell polarity and facilitate T cell activation. First, we will characterize aspects of T cell activation that depend uniquely on ezrin vs those that depend on ERM proteins generally. For these studies, we will use mice with conditional deletion of ezrin, together with shRNA for moesin and a dominant negative mutant that perturbs function of all ERM proteins. In addition, we will study the regulation of ezrin function by tyrosine phosphorylation. Second, we will ask how the DPC functions during T cell signaling, by focusing on two key DPC components: SHP-1 and PI3K. The interactions of each of these proteins with ERM proteins and other DPC components will be explored, and we will test the effects of disrupting DPC organization on their respective downstream signaling pathways. Third, we will study how the DPC functions to define T cell polarity via scribble and hDLG. We will analyze cell polarity in scribble- and hDLG-deficient T cells and compare results with ERM-disrupted T cells. Structure-function analysis of scribble and hDLG will be conducted, and interactions between hDLG and ezrin probed biochemically. Finally, video microscopy will be performed to order the function of hDLG, scribble and ERM proteins in organizing T cell polarity. This project will make extensive use of the scientific cores, and we will draw heavily on the experitise of the other Project leaders in the Program. These studies address the basic biology of T cell activation, and are therefore relevant to the rational development of new vaccines and treatments for immunodeficiency and autoimmune disease. In addition, these studies will be valuable for understanding cancers associated with dysregulation of ezrin and hDLG.